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Compartmentalization of endocannabinoids into lipid rafts in a dorsal root ganglion cell line
Author(s) -
Rimmerman N,
Hughes H V,
Bradshaw H B,
Pazos M X,
Mackie K,
Prieto A L,
Walker J M
Publication year - 2008
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0707561
Subject(s) - lipid raft , diacylglycerol lipase , monoacylglycerol lipase , diacylglycerol kinase , raft , endocannabinoid system , compartmentalization (fire protection) , chemistry , biochemistry , 2 arachidonoylglycerol , receptor , microbiology and biotechnology , biology , cannabinoid receptor , signal transduction , protein kinase c , enzyme , copolymer , organic chemistry , agonist , polymer
Background and purpose: N ‐arachidonoyl ethanolamine (AEA) and 2‐arachidonoyl glycerol (2‐AG) are endogenous cannabinoids binding to the cannabinoid receptors CB 1 and CB 2 to modulate neuronal excitability and synaptic transmission in primary afferent neurons. To investigate the compartmentalization of the machinery for AEA and 2‐AG signalling, we studied their partitioning into lipid raft fractions isolated from a dorsal root ganglion X neuroblastoma cell line (F‐11). Experimental approach: F‐11 cells were homogenized and fractionated using a detergent‐free OptiPrep density gradient. All lipids were partially purified from methanolic extracts of the fractions on solid phase cartridges and quantified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Protein distribution was determined by Western blotting. Key results: Under basal conditions, the endogenous cannabinoid AEA was present in both lipid raft and specific non‐lipid raft fractions as was one of its biosynthetic enzymes, NAPE‐PLD. The 2‐AG precursor 1‐stearoyl‐2‐arachidonoyl‐ sn ‐glycerol (DAG), diacylglycerol lipase α (DAGLα), which cleaves DAG to form 2‐AG, and 2‐AG were all co‐localized with lipid raft markers. CB 1 receptors, previously reported to partition into lipid raft fractions, were not detected in F‐11 membranes, but CB 2 receptors were detected at high levels and partitioned into non‐lipid raft fractions. Conclusions and implications: The biochemical machinery for the production of 2‐AG via the putative diacylglycerol pathway is localized within lipid rafts, suggesting that 2‐AG synthesis via DAG occurs within these microdomains. The observed co‐localization of AEA, 2‐AG, and their synthetic enzymes with the reported localization of CB 1 raises the possibility of intrinsic‐autocrine signalling within lipid raft domains and/or retrograde‐paracrine signalling. British Journal of Pharmacology (2008) 153 , 380–389; doi: 10.1038/sj.bjp.0707561 ; published online 29 October 2007